1. Field of the Invention
This invention relates to electrochemical bioreactor systems that can provide electrical enhancement of chemical synthesis and/or fermentations and/or biotransformations when supplied with electricity, that can generate electrical current detectable at a load, and that may used in chemical or biochemical sensing devices. In particular, the invention relates to improved electrodes for electrochemical bioreactor systems that can use electrical energy as a source of reducing power in fermentation or enzymatic reactions and that can use electron mediators and a biocatalyst, such as cells or enzymes, to produce electricity.
2. Description of the Related Art
A biofuel cell is a device that directly converts microbial metabolic power into electricity using electrochemical technology. (See, for example, Allen, “Cellular Electrophysiology”, p. 247-283, In J. R. Norris and D. W. Ribbons (eds.). Methods in Microbiology. Academic Press, New York, 1992; Bennetto, et al. “The Sucrose Fuel Cell: Efficient Biomass Conversion Using A Microbial Catalyst”, Biotechnol. Lett. 7:699-105, 1985; Roller et al., “Electron-Transfer Coupling In Microbial Fuel Cells: 1. Comparison Of Redox-Mediator Reduction Rates And Respiratory Rates Of Bacteria”, J. Chem. Tech. Biotechnol. 34B:3-12, 1984; and Thurston, et al., “Glucose Metabolism In A Microbial Fuel Cell. Stoichiometry Of Product Formation In A Thionine-Mediated Proteus Vulgaris Fuel Cell And Its Relation To Coulombic Yields”. J. Gen. Microbial. 131: 1393-1401, 1985.). Chemical energy is converted to electric energy by coupling the biodegradative oxidation of organic or inorganic substrates to the chemical reduction of an oxidant at the interface between the anode and the cathode (see, Willner et al. “A Biofuel Cell Based On Pyrroloquinoline Quinone And Microperoxidase-11 Monolayer-Functionalized Electrodes”, Bioelectrochem. Bioenerg, 44:209-214, 1998.). Direct electron transfer from microbial cells to electrodes occurs at very low efficiencies (See, Allen, “Cellular Electrophysiology”, p. 247-283, In J. R. Norris and D. W. Ribbons (eds.). Methods in Microbiology. Academic Press, New York, 1992). In microbial fuel cells, two redox couples are required, one for coupling reduction of an electron mediator to bacterial oxidative metabolism, and the other for coupling oxidation of the electron mediator to the reduction of the electron acceptor on the cathode surface where the electron acceptor is regenerated with atmospheric oxygen (see, Ardeleanu, et al., “Electrochemical Conversion In Biofuel Cells Using Clostridium Butyricum Or Staphylococcus Aureus Oxford”, Bioelectrochem. Bioenerg, 11:273-277, 1983; and Delaney, et al., “Electron-Transfer Coupling In Microbial Fuel Cells. 2. Performance Of Fuel Cells Containing Selected Microorganism-Mediator-Substrate Combinations”, Chem. Tech. Biotechnol. 34b:13-27, 1985).
Electron transfer from a microbial electron carrier to an electrode requires an electron mediator (See, Fultz et al., “Mediator Compounds For The Electrochemical Study Of Biological Redox Systems: A Compilation”, Anal. Chim. Acta. 140:1-18, 1982.). Previous studies reported that metabolic reducing power produced by Escherichia coli or Proteus vulgaris was converted to electricity by using mediators such as 2-hydroxy-1,4-naphtoquinone (HNQ) or thionin (see, Tanaka et al., “Effects Of Light On The Electrical Output Of Bioelectrochemical Fuel-Cells Containing Anabaena Varbilis M-2: Mechanisms Of The Post Illumination Burst”, Chem. Tech. Biotechnol. 42:235-240, 1988; and Tanaka et al., “Bioelectrochemical Fuel-Cells Operated By The Cyanobacterium, Anabaena Variabilis”. Chem. Tech. Biotechnol. 35B: 191-197, 1985). Park et al. in “Electrode Reaction Of Desulfovibrio Desulfuricans Modified With Organic Conductive Compounds”, Biotech. Techniq. 11:145-148, 1997 confirmed that viologen dyes (see, Kim et al., “Benzyl Viologen Cation Radical: First Example Of A Perfectly Selective Anion Ionophore Of The Carrier Type”, Biochem. Biophys. Res. Com., 180:11276-1130, 1982; and Morimyo, “Isolation And Characterization Of Methyl Viologen Sensitive Mutants Of Escherichia Coli K-12”, J. Bacteriol. 170:2136-2142, 1988) cross-linked with carbon polymers and absorbed to Desulfovivrio desulfuricans cell membranes can mediate electron transfer to electrodes. Kim et al. in “Direct Electrode Reaction Of Fe(III)-Reducing Bacterium, Shewanella Putrefacians, J. Microbial. Biotechnol., 9:127-13, 1999 showed that Shawella putrefacians, which contains outer-membrane cytochromes able to reduce Fe3+, was electroactive and, that it could grow on lactate as the electron donor with a graphite felt electrode as the electron acceptor in a complex biofuel cell. U.S. Pat. No. 6,270,649 to Zeikus et al. shows that neutral red is an improved electron mediator for either converting electricity into microbial reducing power for enhanced cell growth and production of reduced end-products (see, Park et al., “Microbial Utilization Of Electrically Reduced Neutral Red And The Sole Electron Donor For Growth And Metabolite Production”, Appl. Environ. Microbiol. 65:2912-2917, 1999; and Park et al., “Utilization Of Electrically Reduced Neutral Red By Actinobacillus Succinogenes: Physiological Function Of Neutral Red In Membrane-Driven Fumarate Reduction And Energy Conservation”, J. Bacteriol. 1812:2403-2410, 1999), or converting microbial reducing power into electricity in biofuel cells (see, Park and Zeikus, “Electricity Generation In Microbial Fuel Cells Using Neutral Red And An Electronophore”, Appl. Environ. Microbiol. 66:1292-1297, 2000). Park et al., in “Electricity Production In Biofuel Cell Using Modified Graphite Electrode With Neutral Red”, Biotech. Lett. 22:1301-1304, 2000 showed that binding neutral red to a graphite electrode further enhanced electron transfer efficiency in microbial fuel cells.
The electrical enhancement of fermentations and biotransformations also involves the utilization of an electrode and electron mediator in a bioreactor system which overproduces reduced end products (see, Hongo et al., “Application Of Electro-Energizing Method To L-Glutamic Acid Fermentation”, Agri. Biolio. Chem., 43: 2075-20811 1979; Hongo et al., “Application Of Electro-Energizing Method To L-Glutamic Acid Fermentation”, Agri. Biolio. Chem., 43: 2083-2086, 1979; Kim et al., “Electron Flow Shift In Clostridium Acetobutylicum Fermentation By Electrochemically Introduced Reducing Equivalent” 1988; Park and Zeikus “Utilization Of Electrically Reduced Neutral Red By Actinobacillus Succinogenes: Physiological Function Of Neutral Red In Membrane-Driven Fumarate Reduction And Energy Conservation”, J. Bacteriol. 181: 403-2410, 1999; and Shin et al., “Evaluation Of An Electrochemical Bioreactor System In The Biotransformation Of 6-Bromo-2-Tetralone To 6-Bromo-2-Tetralol”, Appl Microbiol Biotechnol., DOI 10.1007/s002530100809. Online publication: Sep. 22, 2001.) For example, a graphite felt electrode and soluble neutral red can greatly enhance the yields of succinate produced by fermentation (see Park and Zeikus “Utilization Of Electrically Reduced Neutral Red By Actinobacillus Succinogenes: Physiological Function Of Neutral Red In Membrane-Driven Fumarate Reduction And Energy Conservation”, J. Bacteriol. 181: 403-2410, 1999) and, tetralol produced by yeast transformation (Shin et al., “Evaluation Of An Electrochemical Bioreactor System In The Biotransformation Of 6-Bromo-2-Tetralone To 6-Bromo-2-Tetralol”, Appl Microbiol Biotechnol., DOI 10.1007/s002530100809. Online publication: Sep. 22, 2001). Neutral red works in part by direct chemical reduction of pyridine nucleotides (Park and Zeikus “Utilization Of Electrically Reduced Neutral Red By Actinobacillus Succinogenes: Physiological Function Of Neutral Red In Membrane-Driven Fumarate Reduction And Energy Conservation”, J. Bacteriol. 181: 403-2410, 1999).
The use of oxidoreductases in microbial electrochemical cells has also been proposed. One major factor limiting the utilization of oxidoreductases in chemical syntheses (see, e.g., S. M. Roberts et al., Chimicaoggi, “Some Recent Advances In The Synthesis Of Optically Pure Fine Chemicals Using Enzyme-Catalyzed Reactions In The Key Step”, July/August 1993, pp. 93-104; and D. Miyawaki et al., “Electrochemical Bioreactor With Immobilized Glucose 6-Phosphate Dehydrogenase On The Rotation Graphite Disc Electrode Modified With Phenazine Methosulfate”, Enzg. Microbiol. Technol. 15:525-29, 1993) or in chemical detection, i.e., biosensors (see, e.g., P. N. Bartlett, “Modified Electrode Surface In Amperometric Biosensors”, Med. and Biol. Eng. and Comput. 28: B10-B7, 1990; and D. Miyawaki et al., supra) is the lack of a facile system for regeneration or recycling of the electron transferring cofactors (e.g., nicotinamide adenine dinucleotide, quinones, flavin adenine dinucleotide, etc).
It has been reported by Park and Zeikus in “Utilization Of Electrically Reduced Neutral Red By Actinobacillus Succinogenes: Physiological Function Of Neutral Red In Membrane-Driven Fumarate Reduction And Energy Conversion”, J. Bacteriol. 181:2403-2410, 1999 that neutral red would undergo reversible chemical oxidoreduction with nicotinamide adenine dinucleotide (i.e., recycle nicotinamide adenine dinucleotide electrochemically). It has also been reported that by using soluble neutral red in electrochemical reactors containing microbes that: (1) microbes could grow on electricity alone; (2) diverse microbes could over-produce a variety of reduced biochemicals during fermentations of biotransformations; and (3) microbes could generate electricity during digestion of organic matter. (See, e.g., Park et al., “Microbial Utilization Of Electrically Reduced Neutral Red In The Sole Electron Donor For Growth And Metabolite Production”, Appl. Environ. Microbiol. pp. 2912-2917, 1990; Park and Zeikus, “Electricity Generation In Microbial Fuel Cells Using Neutral Red As An Electronophore”, Appl. Environ. Microbiol., 66:1292-1297, 2000; and U.S. Pat. No. 6,270,649).
Because of the importance of electrodes and electron mediators in bioreactor systems for electricity generation, chemical sensing, and electrical enhancement of chemical synthesis, fermentations and biotransformations, there is a continuing general need for improved electrodes that enhance the rate of electron transfer from cells. Preferably, the improved electrode compositions for increased electron transfer efficiency can use resting cells from pure and mixed bacterial cultures. In one specific application, there is a need for an improved electrode that has utility as an enzymatic fuel cell, as a sensor for succinate detection, and as an engineered catalyst for the synthesis of fumarate or succinate. In particular, there is a need for an enzyme immobilization protocol to link nicotinamide adenine dinucleotide (NAD), neutral red (NR), and fumarate reductase to an electrode in an electrochemical reactor.
Microbial electrochemical cells have previously used two-compartment systems whereby the aerated cathode compartment contains a chemical solution of ferric cyanide and oxygen, and the anode compartment contains bacterial cells, electron mediator, and reduced substrate (see, for example, Ardeleanu, et al., “Electrochemical Conversion In Biofuel Cells Using Clostridium Butyricum Or Staphylococcus Aureus Oxford”, Bioelectrochem. Bioenerg, 11:273-277, 1983; and Park and Zeikus, “Electricity Generation In Microbial Fuel Cells Using Neutral Red And An Electronophore”, Appl. Environ. Microbiol. 66:1292-1297, 2000). Two compartment fuel cells are generally not practical because of the requirement for a ferricyanide solution and aeration in the cathode compartment. Thus, there is also a need for a single compartment microbial electrochemical cell that eliminates the requirements for a ferricyanide solution and aeration in the cathode compartment.